1. Field of the Invention.
The present invention relates to the area of affinity purification of macromolecules. More particularly, the invention provides an affinity membrane, wherein the pore size is based upon the size of the porogen selected, a method for preparation of the membrane, and a method for affinity purification of macromolecules.
2. Description of Related Art.
Affinity membrane filtration (AMF) has recently emerged as an alternative to affinity column chromatography. An advantage of AMF is that high flow rates at low pressure drops can be achieved, thereby greatly improving the washing, elution, and regeneration processes, and decreasing the probability of deactivation of the biomolecules by shortening their exposure to an unfavorable medium.
The key to efficient AMF is the preparation of the affinity membranes. In general, two approaches have been employed to prepare affinity membranes. In the most common method, microporous affinity membranes are prepared from polyethylene, polypropylene, nylon, polysulfone, and glass. However, these membranes are usually hydrophobic and relatively inert, and hence require modifications. In addition, some of the membranes may require amplification of the number of active groups. To overcome these drawbacks, a second approach has been employed wherein membranes are prepared that have preincorporated functional groups. However, the problems with this type of membranes include hydrophobicity (poly glycidyl methacrylate-co-ethylene dimethacrylate membrane), brittleness, and solubility in acids (cellulose acetate membrane). Another drawback with both of the above methods is that the pore size of the membrane cannot be easily controlled.
Recently chitosan membranes have been suggested as affinity membranes for immobilization of various macromolecules having affinity for chitosan. Next to cellulose, chitin (poly (N-acetyl-D-glucosamine)), is the most abundant biopolymer. Chitosan, the deacetylated form of chitin, is soluble in dilute aqueous organic acids but is insoluble in alkaline solutions. Chitosan molecules contain a large number of reactive hydroxyl and amine groups, which can easily attach ligands. In view of its hydrophilicity, excellent film-forming ability, good mechanical properties, and high chemical reactivity (containing hydroxyl and amine groups), chitosan can be an excellent candidate for filtration membranes. Moreover, since chitosan has a positive charge due to the presence of --NH.sub.2 groups, it can be used to selectively adsorb malignant leukemia cells which carry a higher negative charge on their surface than normal cells. Since chitin contains N-acetyl-D-glucosamine units in its structure which can bind certain molecules, it can be employed for affinity purification without further chemical modification. Other advantages of chitosan and chitin are that they are easily available and inexpensive. Moreover, chitin and crosslinked chitosan are insoluble in both acidic and alkaline media making them suitable as filtration membranes.
In addition to affinity filtration, other uses of chitosan membranes include reverse osmosis (Yang and Zall, 1984 J. Food Sci., vol 49:91-93), pervaporation (Tsugita et al., U.S. Pat. No. 4,983,304; Zeng et al., 1993 Membr. Sci. Technol., vol 13:29-32; Goto et al., 1994 Sep. Sci. Technol. vol 29:1915-23), ultrafiltration (Beppu et al., 1993 Kobunshi Ronbunshu vol 50:35-40), and affinity filtration (Zeng and Ruckenstein 1996 J. Membr. Sci. vol 117:271-278). U.S. Pat. No. 5,116,747 to Moo-Young et al. describes the use of a semi-permeable membrane, formed by chitosan and a water soluble polymer, for immobilization of biologically active material. U.S. Pat. No. 5,006,255 to Uragami describes a selective permeable membrane prepared by cross-linking of chitosan by aldehyde, and used for separation of water-alcohol solution.
Currently, there is no suitable method available for the preparation of microporous or macroporous chitosan membranes wherein the size of the pores can be controlled. The most common method to prepare microporous chitosan membranes is the phase-inversion process, using a large molecular weight organic compound as a porogen. The process involves three steps: (1) casting of a solution of the membrane containing a porogen and partial evaporation of the solvent; (2) sol-gel transformation and generation of pores via the addition of a solvent for the porogen; and (3) heat treatment for stabilizing the pore structure and improving the mechanical properties. This method requires rigorous control of various parameters, particularly the kind and amount of porogen and evaporation conditions (time, humidity and temperature). Generally, the porogens employed in the phase-inversion methods for preparing hydrophobic membranes were organic compounds of low molecular weight such as acetone, dimethyl formamide, dimethyl sulfoxide, benzene, etc. To obtain large pores in chitosan membranes, the relatively large molecule of poly(ethylene glycol), molecular weight 35,000, was used as porogen (Zeng and Ruckenstein, 1996 J. Membr. Sci. vol 117:271-278). Although relatively high permeability membranes were obtained, their mechanical properties were not satisfactory, and they had to be placed on another support.
So far, microporous or macroporous chitin membranes have not been available, primarily because no suitable solvent and porogen could be found. A few solvents, such as the mixtures trichloroacetic acid-chloral hydrate-dichloromethane (Brine and Austin, 1975 ACS Symposium Series, Church T. D., Eds., American Chemical Soc., vol 18, p505), dimethylacetamide (DMAc)-LiCl (Rutherford and Austin, 1977 Proc. of the First International Conf. on Chitin and Chitosan, Muzzaralli, R. A. A., Priser, E. R., Eds., MIT Sea Grant Program, Cambridge), and N-methyl-2-pyrrolidone-DMAc-LiCl (Uragami et al., 1981 Polym., vol 30:1155-1156) have been tried. However, it was either almost impossible to completely dissolve chitin in these solvents, or required a long time, followed frequently by degradation.